The major advantage of confocal and multi-photon laser scanning microscopy over conventional epifluorescence microscopy is the capability to perform optical sectioning. To obtain images at reasonable frame rate, however, the excitation intensity of laser scanning microscopy often has to be several-fold stronger than that of epifluorescence microscopy. This is due to the extremely short dwell time per pixel in scanning microscopy. Consequently, for confocal microscopy, significant photo-toxicity can occur in scanned live organisms if a fast time-lapse microscopy is required. (See, e.g. J. B. Pawley, Handbook of biological confocal microscopy, Springer, New York, N.Y., third edition (2006), the disclosure of which is incorporated herein by reference.) On the other hand, multi-photon microscopy has been reported to induce lower photo-toxicity, but it has also been shown to introduce thermal mechanical damage to live tissues through the single-photon absorption of infrared excitation. (See, e.g., S. Potter, Current Biology 6(12), 1595-1598 (1996); M. Cahalan, et al., Nature Reviews Immunology 2(11), 872-880 (2002); and B. Masters, et al., Journal of Biomedical Optics 9(6), 1265-1270 (2004), the disclosures of which are incorporated herein by reference.)
One potential way to resolve photo- and thermo-damage in scanning microscopy and gain speed for image acquisition is to implement the capability of optical sectioning in wide-field microscopy. Several methods have been proposed, such as light-sheet illumination microscopy and structured light microscopy [6]. (See, e.g., J. Huisken, et al., Science 305(5686), 1007-1009 (2004); and M. Neil, et al., Optics Letters 22(24), 1905-1907 (1997), the disclosures of each of which are incorporated herein by reference.) Both methods are technically complicated due to the requirement of additional mechanical parts that synchronize with axial scanning components. For example, light-sheet microscopy obtains optical sectioning by illuminating the sample from lateral side; this introduces mechanical complexity into the optical system as well as makes the preparation of samples difficult. In addition, structured light microscopy needs to take multiple images and retrieve the axial resolution by computing the signal root mean square at each pixel. This reduces the image acquisition rate and is an inefficient use of the quantum yield of the fluorophores.
Recently, Oron et al. have developed a technique, referred to as temporal focusing, to obtain optical sectioning in a wide-field setup of multi-photon microscopy. (See, D. Oron, et al., Opt Express 13(5), 1468-76 (2005), the disclosure of which is incorporated herein by reference.) In their study, temporal focusing was achieved using a blazed diffraction grating as a scatterer. In such a technique, different wavelengths of light re-gain their coherence only when they meet at the image plane, thereby creating the optical sectioning. However, the signal level is several orders of magnitude weaker than that can be achieved by conventional scanning microscopy, due to the inevitable reduction of multi-photon excitation efficiency in the setup. This leads to a significant reduction of the image acquisition rate even for tissues stained with fluorescent dye, which is usually much brighter than fluorescent protein expressed in live tissues. In Oron et al.'s work, the frame rate is roughly 0.033 frames per second (fps) at cells stained with DAPI (a bright dye for chromosome staining). In addition, the core component in current temporal focusing setup, a blazed diffraction grating, is fabricated for a specific wavelength window each. This increases the systems complexity when two or more excitation wavelengths are required for the application, which is common in most biomedical studies.
Accordingly, a need exists to find a novel approach to perform multi-photon microscopy that reduces system complexity and photo-toxicity, while maintaining the spatial resolution, frame rate and other optical advantages of the technique.